The Doa4 deubiquitinating enzyme is required for ubiquitin homeostasis in yeast

Attachment of ubiquitin to cellular proteins frequently targets them to the 26S proteasome for degradation. in addition, ubiquitination of cell surfac e proteins stimulates their endocytosis and eventual degradation in the vac uole or lysosome. In the yeast Saccharomyces cerevisiae, ubiquitin is a lon g-lived protein, so it must be efficiently recycled from the proteolytic in termediates to which it becomes linked. We identified previously a yeast de ubiquitinating enzyme, Doa4, that plays a central role in ubiquitin-depende nt proteolysis by the proteasome. Biochemical and genetic data suggest that Doa4 action is closely linked to that of the proteasome. Here we provide e vidence that Doa4 is required for recycling ubiquitin from ubiquitinated su bstrates targeted to the proteasome and, surprisingly, to the vacuole as we ll. In the doa4 Delta mutant, ubiquitin is strongly depleted under certain conditions, most notably as cells approach stationary phase. Ubiquitin depl etion precedes a striking loss of cell viability in stationary phase doa4 D elta cells. This loss of viability and several other defects of doa4 Delta cells are rescued by provision of additional ubiquitin. Ubiquitin becomes d epleted in the mutant because it is degraded much more rapidly than in wild -type cells. Aberrant ubiquitin degradation can be partially suppressed by mutation of the proteasome or by inactivation of vacuolar proteolysis or en docytosis. We propose that Doa4 helps recycle ubiquitin from both proteasom e-bound ubiquitinated intermediates and membrane proteins destined for dest ruction in the vacuole.